The present invention refers to the general field of the technology of the DNA recombinant proteins, for the production of the gp90 envelope protein of Equine Infectious Anemia virus (EIAV), to be used in diagnosis, vaccination, antibody production or in research field.
The equine infectious anemia (EIA) is one of the oldest diseases caused by virus, having been described for the first time in France by LIGNEE, Rec. Med. Vet, 20:30, 1843, and recognized as viral disease by VALLEE and CARRE. Acad. Sci., 139:331-333, 1904. The disease affects exclusively the members of the family Equidae presenting a worldwide distribution and consequently of great economical importance.
The EIA virus (EIAV) is classified as a lentivirus of the Retroviridae family (CHARMAN et al. J. Virol. 19(2):1073-1076, 1976), it is genetic and antigenically related to the other lentiviruses which are characterized by causing persistent infection. The EIA has played a specially important role in comparative virology and in the studies of the acquired immunodeficiency syndrome (AIDS). Besides their morphological identity, both viruses possess similarities in terms of nucleotide sequences that code for structural proteins, and they infect the same cells. These viruses present genetic and antigenic variants during persistent infections, which is associated to the immunologic evasion MONTAGNIER et al. Ann. Virol., 135:119-134, 1984, MONTELARO et al. J. Biol. Chem., 259:10539-10544, 1984, RUSHLOW et al. Virology, 155:309-321, 1986, STREICHER et al. J. Am. Med. Assoc. 256:2390-2391 1986, STOLER et al. J. Am. Med. Assoc. 256,2360-2364, 1986 and HAHN
The transmission of EIAV occurs mainly by arthropods vectors (tabanideos) by inoculating the virus into the animal""s blood stream when feeding themselves (mechanical transmission) justifying the high prevalence of EIA in hot areas favorable to the life cycle of of these vectors ISSEL et al. Vet. 17:251-286, 1988. EIA can also be transmitted by the placenta and colostro of mare with high virus titers, and by needles and surgical instruments contaminated with blood COGGINS Comparative diagnosis of viral diseases, N.Y., 4:646-658, 1981. The disease present the acute forms, subacute, chronic and mainly inaparent or assimptomaticxn lSSEL and COGGINS, J. Am. Vet. Med. Assoc. 174(7):727-33, 1979, and the most prominent signs are the feverish episodes, anemia hemolitica, anorexia, fast weight loss and ventral edema.
Considering the high prevalence of assymptomatic carriers, the non conclusive clinical diagnosis and the possibility to confuse with other diseases as the trypanosomiases, piroplasmose, leptospirose, hepatitis and endoparasitoses the laboratory diagnosis plays a decisive role in the control and prevention of EIA.
The accepted way to diagnose the presence of EIA has been to detect the presence of antibodies specific for the disease in the serum of affected animals using the Coggins or agar gel diffusion test described in U.S. Pat. No. 3,929,982 and U.S. Pat. No. 3,932,601. In the Coggins test, a prepared antigen is placed alongside the senum to be tested in an agar or gel medium. If EIA antibodies are present in the test serum, they will diffuse toward the antigen forming a precipitin line in the agar medium where they eventually meet. The antigen is prepared, using spleen of infected horses COGGINS and NORCROSS Cornell. Vet. 60(2):330-5, 1970 or in culture of horse leucocytes NAKAJIMA and USHIMI Infect Immun, 3(3):373-7, 1971.
This methodology is inherently insensitive in that the EIA antigen may be contaminated with non-EIA antigens during its preparation. Antibodies against non-EIA antigens may be present in the test serum and can react Even if the prepared EIA viral antigen can be purified, the Coggins test is labor intensive and demanding of considerable expertise in interpretation of results. The Coggins test procedure is also slow to yield results, it takes twenty-four to forty-eight hours for the formation of clearly visible precipiting lines.
Porter, U.S. Pat. No. 4,806,467, discloses a method for detecting the EIA virus using a complete enzyme-linked immunoabsorbent assay incorporating a purified viral antigen and a monoclonal antibody. To obtain the antigen, the EIA virus must first be cultured. The antigen is the p26 core protein of the EIA virus and is obtained through (purification of the cultured virus by a variety of means) well known in the art. The technique of culturing a virus increases the likelihood that the assay will yeild false positive results since the virus may be contaminated with other forms of protein. Addtionally, the EIA virus is hard to culture, making the Porter approach difficult for large scale production.
The use of a synthetic peptide in an enzyme linked (immunosorbent assay) for the detection of human immunodeficiency virus (HIV) is disclosed in Shoeman, R. L. et al, Analytical Biochemistry 161:370-379 (1987). HIV and the EIA virus are members of the retrovirus family but have dissimilar structures and distinct amino acid sequences.
The main component of these preparations is therefore a protein of the virai capsid whose molecular weight is 26 KDa, (denominated p26) This is the most abundant protein of the viral particle PAREKH et al. Virology, 107:520-525, 1980, GELDERBLON, AIDS 5:617-638, 1991, and it is highly conserved within the variant samples of the isolated viruses HUSSAIN et al. J. Virol. 61:2956-2961, 1987, SALINOVICH et al. J. Virol. 57:71-80, 1986. and infected horses present specific antibodies anti-p26.
Darrel and Peisheng, the U.S. Pat. No. 5.427,907, discloses a method to use a synthetic peptide as the antigen in an immunoassay for the detection of antibodies against the equine infectious anemia virus in the serum of horses. This procedure include only the search of some epitopes of virus proteins.
It is an object of the present invention to describe the recombinant gp90 envelope protein from AIEV, their corresponding encoding recombinant DNA molecule and the process of production of the recombinant gp90 envelope protein produced through techniques of genetic engineering, to be used for diagnosis, vaccination or in research.